Epigenetic disruptions of gene expression such as by DNA methylation and histone modifications are profoundly involved in tumorigenesis. For leukemia, the gene hypermethylated in cancer 1 (“HIC1”) is unique because hypermethylation of the gene's promoter region occurs progressively towards the late phases of hematologic malignancies. HIC1 encodes a DNA-binding, zinc finger transcriptional factor that is essential for mammalian development. The HIC1 gene is inactivated but not mutated in certain human cancers such as chronic myelogeneous leukemia (CML) and relapsed acute lymphocytic leukemias following chemotherapy. Using mouse genetics, the importance of HIC1 in of tumorigenesis has recently been demonstrated. Germline disruption of one copy of HIC1 predisposes mice to a late on-set and gender-dependent spectrum of malignant tumors wherein promoter hypermethylation of the wild type HIC1 allele is associated with loss of function of this gene. It is also known that HIC1 plays a synergistic role with p53 in suppressing the development of age-dependent cancers. Germline disruption of one copy each of HIC1 and p53 on opposite (trans) chromosomes or same (cis) chromosomes in mice results in altered tumor spectrum, earlier appearance and increased prevalence and aggressiveness of osteosarcomas. Indeed, a low frequency of blast crisis megakaryocytic leukemia is found in cis HIC1 and p53 double heterozygous mice. See FIG. 1.
A key mechanism by which HIC1 suppresses tumorigenesis is through its regulation of the stress and DNA damage responsive gene, SIRT. SIRT1 is a mammalian orthologue of yeast silent information regulator 2 (Sir2) that is required for yeast lifespan extension upon calorie restriction (Lin et al., 2000). An extra copy of Sir2 extends life span in yeast, fly and worm. SIRT1 is a class III histone deacetylase whose enzymatic activity is dependent on cofactor NAD. SIRT1 is insensitive to histone deacetylase inhibitor trichostatin A (TSA) which inhibits class I and II deacetylases. SIRT1 is involved in regulation of a variety of cellular functions including survival, glucose homeostasis and fat metabolism through deacetylating histones and non-histone proteins (Saunders & Verdin 2007). SIRT1 levels increase in response to metabolic stresses such as calorie restriction and nutrient starvation (Cohen et al. 2004; Nemoto et al. 2004). The mammalian SIRT1 promotes cell survival under oxidative stress, genotoxic stress and DNA damage through multiple substrates including p53 (Luo et al. 2001; Vaziri et al. 2001), Ku70 (Cohen et al. 2004) and FOXO proteins. For example, SIRT1 promotes cell survival using p53 via a pathway that includes deacetylation of p53 and attenuation of its ability to activate downstream targets to control apoptosis. HIC1 forms a complex with SIRT1 protein. This HIC1/SIRT1 protein complex directly binds to the SIRT1 promoter in vivo to repress SIRT1 gene transcription. Loss of HIC1 expression by promoter hypermethylation upregulates SIRT1 in cancer cells, attenuates p53 activity by deacetylation and allows cells to bypass apoptosis and survive stress and DNA damage. Inhibition of SIRT1 function in cells without HIC1 abolishes the resistance to apoptosis.
Chronic myelogenous leukemia (CML) is a fatal hematopoietic disorder resulting from malignant transformation of bone marrow progenitor cells. The disease progresses from chronic phase, to accelerated phase, to terminal blast crisis phase. CML is characterized by a reciprocal translocation of chromosome 9 and 22 that creates an oncogenic fusion gene, BCR-ABL. This gene produces a protein with deregulated BCR-ABL tyrosine kinase activity. Imatinib mesylate (also known as imitanib, Gleevac or STI-571) is a potent ABL tyrosine kinase inhibitor. In most chronic phase patients, treatment with imatinib results in complete cytogenic responses (CCR) or remission and infrequent relapse. However, in most blast crisis patients, there is a poor response to imatinib treatment and a high frequency of relapse in those patients having an initial response. The molecular mechanisms of the resistance to imatinib may consist of both BCR-ABL dependent and independent pathways. BCR-ABL dependent pathways are characterized by genetic alterations of the BCR-ABL gene.
The clinical resistance to imatinib treatment is mediated primarily by genetic mutations of the BCR-ABL kinase domain, and to a lesser extent, by amplification of the BCR-ABL gene. In relapsed CML patients, more than 15 BCR-ABL mutations have been identified. These mutations confer various degrees of resistance to imatinib. Mechanisms for formation of BCR-ABL mutations in CML are not clear. The vast majority of BCR-ABL mutations are detected in relapsed patients, but pre-existing mutations including a T315I mutation are also found in patients before imatinib treatment (Gorre et al., 2001; Shah et al., 2002). The T315I mutation has been identified thus far as being frequent and the most resistant mutation. Located in the center of the imatinib binding site is Thr315 and the T315I mutation blocks the drug from binding to the ABL kinase. In addition, imatinib suppresses proliferation of human CML leukemic progenifor cells, but cannot eliminate them in vivo. Thus, most subjects in CCR or remission continue to harbor residual leukemia cells (Bhatia et al. 2003). Similarly, imatinib treatment results in remission of CML in mouse models of the disease, but fails to eliminate leukemic stem cells (Hu et al. 2006; Neering et al. 2007). Because of this, in vitro studies of the process by which BCR-ABL is mutated in CML cells is difficult because, unlike what occurs in vivo, nearly all CML cell lines derived from blast crisis CML are sensitive to 1 μM STI-571 treatment. (Deininger et al. 1997). Nilotinib (AMN107) is a recently developed BCR-ABL inhibitor having greater potency. It inhibits most of the known mutants with the exception of the T315I mutation. Similarly, the potent dual SRC-ABL kinase inhibitor dasatinib (BMS-354825) inhibits 14 of 15 BCR-ABL mutants but not T315I. However, in vivo, CML patients with a T315I mutation do not respond to either nilotinib or dasatinib. Without further effective treatment, these blast crisis patients are terminal. Accordingly, a method of treating these relapsed patients or preventing formation of this resistant mutation is highly desired.
Several resistant CML cell lines have been developed by gradually exposing cells to increasing concentrations of STI-571 (Mahon et al., 2000). However, these resistant cell lines all have BCR-ABL gene amplification but lack mutations. This is opposite to the results seen in patients. Today, most in vitro mutation studies are carried out using murine cell lines such as Ba/F3 cells, a murine pro-B cell line transfected with genetically engineered BCR-ABL mutations. (La Rosee et al. 2002; Shah et al., 2004; von Bubnoff et al., 2006; von Bubnoff et al., 2005; Weisberg et al., 2005). Although these cell lines are important for addressing mutant kinase activity, they do not reflect in vivo mechanisms of BCR-ABL mutagenesis, and thereby cannot be used to address mechanisms of BCR-ABL mutagenesis in natural cellular and molecular contexts of CML, and cannot be applied to development of strategies for preventing such mutations. The use of these cells also excludes the possibility of studying other genetic and epigenetic alterations accompanying the BCR-ABL mutagenesis process in mutant CML cells. Thus, a CML cell line having one or more BCR-ABL mutations is also highly desired because it is useful as a model system for CML disease. Use of such cell lines will facilitate further study of the mechanism of disease development and progression and assist in the further identification of therapeutic treatments for this disease.